The Breeding Of Xylanase-producing Marine Microorganism And Research On Enzymatic Properties

Xylanases are enzymes that catalyze the hydrolysis of xylan, which are widely used in feed, food, paper-making and alcohol industries. Xylanases derive from various sources, but mainly from earth microorganism rather than marine microorganism. This study screened strain B659 with great xylanase-producing ability from different marine samples. The positive mutant strain Wl-40 was obtained through mutation breeding and its fermentation optimization and enzymatic properties were researched. The results are as follows:1. With self-made bagasse xylan chosen as the only carbon source of primary screening medium, totally 74 strains which showed transparent zones and different morphology were screened from water and mud samples of Pingtan Sea, Fuzhou, mud sample of Dasubu Lake, songyuan, mud sample and algaes of Sandu’ao Sea, Ningde, abalone visceral and arctic mud sample. The shake flask fermentation results showed that 45 strains had xylanase-producing capabilities and strain B659 was highest of them with the xylanase activity of 525.3U/mL. Via morphological characteristics and 16s rDNA sequence analysis, strain B659 was identified as Bacillus stratosphericus.2. The stable mutant strain G3-17 with 13.9% enhancement of xylanase activity was screened from the UV mutagenesis of strain B659. A stable mutant strain W1-40 with 11.6% xylanase activity increase was obtained by microwave mutation and screening from strain G3-17. In 72h fermentation of strain B659 and strain W1-40, the xylanase activity of latter (645.2U/mL) achieved 24.6% elevation than the former(517.9U/mL).3. The xylanase-producing medium and fermentation condition optimization of strain W1-40 suggested the optimized culture medium contained 6%(w/v) wheat bran, 1%(w/v) self-made bagasse xylan, 1%(w/v) bean cake powder, 10mmol/L K2HPO4, 0.03%(w/v) Tween-80, with initial culture pH 6.0. The fermentation should maintain at 30℃ and 230r/min with 8%(v/v) inoculation in 25mL/250mL liquid volumn. After optimization, the xylanase activity reached 2838.3U/mL, which raised 3.4 times than before and shortened period from 72h to 30h.4. The enzymatic properties of xylanase from strain W1-40 was studied. The results indicated that the optimal temperature and pH for reaction was 55℃ and 6.5. Its great alkali resistance made it retained 78.3% activity after 2h incubation at 37℃ and pH 8.0-10.0.10 mmol/L K+ and 1 mmol/L Fe2+ activated xylanase activity while Mn2+, Cu2+ and Co2+ inhibit it obviously. The test of specific substrate indicated that xylan from beechwood was the optimum substrate for the xylanase with slightly cellulose activity. The kinetic reaction experiments showed that Km value was 4.57 mg/mL and Vmax was 1886.8 U/mL.